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D M Irwin

Publications and source records attributed to D M Irwin.

At least 37 records · Page 2Linked to original sources
PubMed1996

Molecular evolution of ruminant lysozymes.

The evolution of a new digestive enzyme, stomach lysozyme, from an antibacterial host defense enzyme provides a link between molecular evolution and organismal evolution. Lysozymes have been recruited at least three times (twice from a conventional lysozyme c and once from a calcium-binding lysozyme c) in vertebrates for functioning in the stomach. The recruitment of lysozyme for its new biological function involved many molecular changes, beyond those required to adapt the protein to function in the stomach. The evolution of the stomach lysozyme gene has been extensively studied in ruminant artiodactyls. In ruminants, the lysozyme c gene has duplicated to yield a family of about ten genes. These duplications allowed: (1) specialization of gene function and (2) increased levels of expression. The ruminant stomach lysozyme genes have evolved in an episodic fashion - there was a period of rapid adaptive sequence evolution, driven by positive selection in the early ruminant, that was followed by an increase in purifying selection upon the well-adapted stomach lysozyme sequence among modern species. Recombination of small portions (exons) of the genes between members of the lysozyme gene family may have aided in adaptive evolution. Evolution to a stomach lysozyme is not irreversible; at least one member of the ruminant stomach lysozyme gene family appears to have reverted to a more ancestral function, yet retains hallmarks of its history as a stomach lysozyme.

Animals↗
PubMed1995

Trout and chicken proglucagon: alternative splicing generates mRNA transcripts encoding glucagon-like peptide 2.

In mammals, the proglucagon gene is transcribed into a single identical mRNA in pancreas, intestine, and brain. The proglucagon mRNA encodes glucagon and two glucagon-like peptides (GLP 1 and GLP 2), whose production is regulated by tissue-specific proteolytic processing. Previously characterized pancreatic proglucagon cDNAs from birds and fish encode glucagon and only one glucagon-like peptide, GLP 1. The isolation of intestinal proglucagon cDNAs from the rainbow trout, Oncorhynchus mykiss, and chicken, Gallus gallus, shows that the proglucagon gene of fish and birds also contains the sequence of a second glucagon-like peptide, GLP 2. In contrast to the proglucagon mRNAs from mammals, fish and bird proglucagon mRNAs from pancreas and intestine have different 3'-ends that are due to alternative mRNA splicing. The intestinal mRNA was found to be spliced to one or more exons, which encode GLP 2, while the pancreatic mRNA terminates within the intron between the exons encoding GLP 1 and GLP 2. These results show that proglucagon gene expression is regulated at the level of mRNA splicing and serve to reemphasize the potential biological importance of GLP 2.

Alternative Splicing↗
PubMed1995

Evolution of the bovine lysozyme gene family: changes in gene expression and reversion of function.

Recruitment of lysozyme to a digestive function in ruminant artiodactyls is associated with amplification of the gene. At least four of the approximately ten genes are expressed in the stomach, and several are expressed in nonstomach tissues. Characterization of additional lysozymelike sequences in the bovine genome has identified most, if not all, of the members of this gene family. There are at least six stomachlike lysozyme genes, two of which are pseudogenes. The stomach lysozyme pseudogenes show a pattern of concerted evolution similar to that of the functional stomach genes. At least four nonstomach lysozyme genes exist. The nonstomach lysozyme genes are not monophyletic. A gene encoding a tracheal lysozyme was isolated, and the stomach lysozyme of advanced ruminants was found to be more closely related to the tracheal lysozyme than to the stomach lysozyme of the camel or other nonstomach lysozyme genes of ruminants. The tracheal lysozyme shares with stomach lysozymes of advanced ruminants the deletion of amino acid 103, and several other adaptive sequence characteristics of stomach lysozymes. I suggest here that tracheal lysozyme has reverted from a functional stomach lysozyme. Tracheal lysozyme then represents a second instance of a change in lysozyme gene expression and function within ruminants.

Animals↗
PubMed1994

Evolution of prothrombin: isolation and characterization of the cDNAs encoding chicken and hagfish prothrombin.

The cDNA sequences of chicken and hagfish prothrombin have been determined. The sequences predict that prothrombin from both species is synthesized as a prepro-protein consisting of a putative Gla domain, two kringle domains, and a two-chain protease domain. Chicken and hagfish prothrombin share 51.6% amino acid sequence identity (313/627 residues). Both chicken and hagfish prothrombin are structurally very similar to human, bovine, rat, and mouse prothrombin and all six species share 41% amino acid sequence identity. Amino acid sequence alignments of human, bovine, rat, mouse, chicken, and hagfish prothrombin suggest that the thrombin B-chain and the propeptide-Gla domain are the regions most constrained for the common function(s) of vertebrate prothrombins.

Amino Acid Sequence↗
PubMed1993

Multiple cDNA sequences of bovine tracheal lysozyme.

The principal role of lysozyme is to prevent bacterial invasion at body surfaces. We are interested in how lysozyme is regulated at the surface of the respiratory tract, where the serous gland cell is regarded as the primary cellular source of this enzyme. Since the cow genome contains at least 10 lysozyme-like genes, our objective was to determine which of them are expressed in the cow tracheal gland serous cell. By screening tracheal cDNA libraries with a probe constructed from the cDNA encoding stomach lysozyme 2, we obtained 3 lysozyme cDNAs: 5a (1023 base pairs (bp)), 7a (1060 bp), and 14d (1249 bp). cDNA 7a corresponds to a previously reported gene (showing sequence identity to the stomach 2 lysozyme gene), whereas cDNAs 5a and 14d correspond to lysozyme genes not previously reported. Northern blot analysis of cow tracheal RNA showed lysozyme mRNAs of three distinct lengths. Based on hybridization with probes specific for each cDNA, we determined that the longest transcript corresponded to cDNA 5a, the shortest to 7a, and the intermediate-length transcript to 14d. Cultured cow tracheal gland serous cell RNA, reverse transcribed and amplified by the polymerase chain reaction with primers common to all three cDNAs, yielded a product that hybridized to oligonucleotide probes specific for all three cDNAs but most strongly to that for 5a. These results indicate that multiple lysozyme mRNAs are expressed in the cow trachea and that the lysozyme encoded by cDNA 5a is the major form expressed in the tracheal gland serous cell. This serous cell lysozyme is predicted to differ importantly in structure from both 7a and 14d lysozymes, with an arginine:lysine ratio almost 10-fold higher. The sequence differences may underlie functional differences, including variable resistance to proteolysis and variable affinity for large polyanions (e.g. mucins) found in the respiratory tract lumen.

Amino Acid Sequence↗
PubMed1993

Evolution of rodent lysozymes: isolation and sequence of the rat lysozyme genes.

Mice have two lysozyme c genes that are the products of a gene duplication. The rat genome also possesses two lysozyme genes, which implies that the duplication of the lysozyme gene preceded the mouse-rat divergence. Concerted evolution has homogenized the 3' half (middle of exon 2 through 3' flanking) of the genes in each species. A mosaic pattern of concerted evolution is found for the 5' half of the gene, with both the 5' and 3' ends, but not the middle, of exon 1 evolving in a concerted fashion. Differences exist in the homogenization pattern in the rat and mouse, suggesting the existence of species-specific patterns of concerted evolution. Exon 1 contains most of the amino acid substitutions that distinguish mouse M from mouse P lysozyme or rat 1 from rat 2 lysozyme. Some of the amino acid differences found between mouse lysozyme genes are found also in the rat, and they suggest that rat 1 and 2 are homologous to mouse M and P, respectively. Nuclease S1 mapping indicated that only the rat 1 gene is expressed at detectable levels, and, specifically, that this gene is expressed by cells in the lung, small intestine, and spleen. The mouse expresses different lysozyme genes in the intestinal (P) and macrophage (M) cells, while the rat expresses only one gene in both of these tissues. The absence of detectable expression and the accumulation of a large number of amino acid substitutions in the parts of exons 1 and 2 of the rat 2 gene not participating in concerted evolution suggest that the rat 2 gene is a pseudogene.

Amino Acid Sequence↗
PubMed1993

Characterization of the cow stomach lysozyme genes: repetitive DNA and concerted evolution.

Cow stomach lysozyme genes have evolved in a mosaic pattern. The majority of the intronic and flanking sequences show an amount of sequence difference consistent with divergent evolution since duplication of the genes 40-50 million years ago. In contrast, exons 1, 2, and 4 and immediately adjacent intronic sequences differ little between genes and show evidence of recent concerted evolution. Exon 3 appears to be evolving divergently. The three characterized genes vary from 5.6 to 7.9 kilobases in length. Different distributions of repetitive DNA are found in each gene, which accounts for the majority of length differences between genes. The different distributions of repetitive DNA in each gene suggest the repetitive elements were inserted into each gene after the duplications that give rise to these three genes and provide additional support for divergent evolution for the majority of each gene. The observation that intronic and flanking sequences are evolving divergently suggests that the concerted evolution events involved in homogenizing the coding regions of lysozyme genes involve only one exon at a time. This model of concerted evolution would allow the shuffling of exon-sized pieces of information between genes, a phenomenon that may have aided in the early adaptive evolution of stomach lysozyme.

Animals↗
PubMed1993

Physical mapping of the lysozyme gene family in cattle.

Amplification of an ancestral lysozyme gene in artiodactyls is associated with the evolution of foregut fermentation in the ruminant lineage and has resulted in about ten lysozyme genes in true ruminants. Hybridization of a cow stomach lysozyme 2 cDNA clone to restricted DNAs of a panel of cow x hamster hybrid cell lines revealed that all but one of the multiple bovine-specific bands segregate concordantly with the marker for bovine syntenic group U3 [Chromosome (Chr) 5]. The anomalous band was subsequently mapped to bovine syntenic group U22 (Chr 7) with a second panel of hybrids representing all 31 bovine syntenic groups. By two-dimensional pulsed-field gel electrophoresis the lysozyme genes on cattle Chr 5 were shown to be clustered on a 2- to 3-Mb DNA fragment, while the lactalbumin gene and pseudogenes that are paralogous and syntenic with the lysozymes were outside the lysozyme gene cluster. Chromosomal fluorescence in situ hybridization of a cocktail of lysozyme genomic clones localized the lysozyme gene cluster to cattle Chr 5 band 23, corroborating the somatic cell assignment.

Animals↗
PubMed1993

Chromosomal localization of the lysozyme gene cluster in river buffalo (Bubalus bubalis L.).

Lysozyme (LYZ) is an antibacterial enzyme which allows the digestion of bacteria present in tears and saliva. In the true stomach of ruminants LYZ breaks open the bacteria of the foregut, which are subsequently digested by typical mammalian digestive enzymes, allowing the incorporation of nutrients from the bacteria. Southern analysis with a single exon from a cow lysozyme gene revealed that there are about 10 genes in ruminants (Irwin & Wilson 1989), while pig and primates have a single lysozyme gene (Swanson et al. 1991) and camels have two (Irwin et al. 1992). The higher number of LYZ genes in ruminants is believed to be the result of gene duplication associated with the evolution of foregut fermentation (Irwin et al. 1992). Recently, the genomic organization of the lysozyme gene family has been determined in domestic cattle, and, using a cocktail of genomic clones, the lysozyme gene cluster (LYZ/) was assigned to chromosome (Chr) 5, band 23 by fluorescence in situ hybridization (FISH) (Gallagher et al. 1993). In our continued effort to test the genetic homology of conserved chromosome banding regions between cattle and river buffalo, and to extend the river buffalo physical gene map, we have mapped the LYZ/ by FISH and R-banding.

Animals↗
PubMed1992

Evolutionary genetics of ruminant lysozymes.

Comparative studies of mammalian lysozymes and their genes have contributed to knowledge of how new functions arise during evolution. The recruitment of lysozymes for functioning in the stomach fluid of ruminants has occurred in response to selection pressures that are partly known and on a time-scale that is known. A semiquantitative analysis of adaptive evolution is thus made possible by the ruminant lysozyme system. Large-scale production of lysozyme by the stomach lining entailed gene duplication as well as a change in gene expression. Remoulding of the lysozyme for working and lasting in the stomach fluid involved accelerated amino acid replacements, which may have been facilitated by intergenic recombination. The possibility that multigene families can accelerate adaptive evolution, by virtue of their capacity for bringing together functionally coupled substitutions, receives emphasis in this review.

Adaptation, Physiological↗
PubMed1991

Stomach lysozyme gene of the langur monkey: tests for convergence and positive selection.

Genomic blotting and enzymatic amplification show that the genome of the langur monkey (like that of other primates) contains only a single gene for lysozyme c, in contrast to another group of foregut fermenters, the ruminants, which have a multigene family encoding this protein. Therefore, the langur stomach lysozyme gene has probably evolved recently (i.e., within the period of monkey evolution) from a conventional primate lysozyme. The sequences of cDNAs for the stomach lysozyme of langur and the conventional lysozymes of three other Old World monkeys were determined. Identification of the promoter for the stomach gene and comparison to the human gene, which is expressed conventionally in macrophages, show that both lysozyme genes use the same promoter. This suggests that the difference in expression patterns is due to change(s) in enhancer or silencer regulatory elements. With the cDNA sequences the hypothesis that the langur stomach lysozyme has converged in amino acid sequence upon the stomach lysozymes of ruminants is tested. Consistent with the convergence hypothesis, only those sites that specify amino acids in the mature lysozyme are shared uniquely with ruminant lysozyme genes. None of the silent sites at third positions of codons or in noncoding regions support a link between the langur and ruminants. Statistical analysis based on silent sites rules out the possibility of horizontal transfer of a stomach lysozyme gene between the langur and ruminant lineages and supports the close relationship of the langur lysozyme gene to that of other monkeys.

Animals↗
PubMed1991

Evolution of the cytochrome b gene of mammals.

With the polymerase chain reaction (PCR) and versatile primers that amplify the whole cytochrome b gene (approximately 1140 bp), we obtained 17 complete gene sequences representing three orders of hoofed mammals (ungulates) and dolphins (cetaceans). The fossil record of some ungulate lineages allowed estimation of the evolutionary rates for various components of the cytochrome b DNA and amino acid sequences. The relative rates of substitution at first, second, and third positions within codons are in the ratio 10 to 1 to at least 33. For deep divergences (greater than 5 million years) it appears that both replacements and silent transversions in this mitochondrial gene can be used for phylogenetic inference. Phylogenetic findings include the association of (1) cetaceans, artiodactyls, and perissodactyls to the exclusion of elephants and humans, (2) pronghorn and fallow deer to the exclusion of bovids (i.e., cow, sheep, and goat), (3) sheep and goat to the exclusion of other pecorans (i.e., cow, giraffe, deer, and pronghorn), and (4) advanced ruminants to the exclusion of the chevrotain and other artiodactyls. Comparisons of these cytochrome b sequences support current structure-function models for this membrane-spanning protein. That part of the outer surface which includes the Qo redox center is more constrained than the remainder of the molecule, namely, the transmembrane segments and the surface that protrudes into the mitochondrial matrix. Many of the amino acid replacements within the transmembrane segments are exchanges between hydrophobic residues (especially leucine, isoleucine, and valine). Replacement changes at first and second positions of codons approximate a negative binomial distribution, similar to other protein-coding sequences. At four-fold degenerate positions of codons, the nucleotide substitutions approximate a Poisson distribution, implying that the underlying mutational spectrum is random with respect to position.

Amino Acid Sequence↗
PubMed1990

Concerted evolution of ruminant stomach lysozymes. Characterization of lysozyme cDNA clones from sheep and deer.

Contradictory evolutionary histories of ruminant lysozymes have been predicted by analysis of genomic blots (Irwin, D.M., Sidow, A., White, R., and Wilson, A.C. (1989) in The Immune Response to Structurally Defined Proteins: The Lysozyme Model (Smith-Gill, S.J., and Sercarz, E.E., eds) pp. 73-85, Adenine Press, Guilderland, NY) and sequences of cow stomach lysozyme cDNAs (Irwin, D.M., and Wilson, A.C. (1989) J. Biol. Chem. 264, 11387-11393). Genomic blots indicate that the amplification of the lysozyme gene family occurred 40-50 million years ago, while the cDNA sequences imply that the stomach genes began diverging from one another after the splitting of the deer and cow lineages, 25 million years ago. To resolve this contradiction, we characterized 111 stomach lysozyme cDNAs from two additional ruminant species: domestic sheep and axis deer. The cDNA sequences of the coding region of mature lysozyme together with the 3'-untranslated region were obtained from abomasum (true stomach) mRNA with the use of the polymerase chain reaction. The two primers for amplifying the cDNA were a lysozyme-specific primer, encoding a conserved sequence at the amino terminus of mature stomach lysozyme, and oligo(dT) as a general mRNA primer. Comparison of the cDNA sequences from these species to one another and to those of the cow revealed that different parts of the ruminant stomach lysozyme genes have had different evolutionary histories. The 3'-untranslated region has evolved in a divergent fashion since the original duplications 40-50 million years ago, supporting the genomic blot interpretation; by contrast, the coding region has evolved in a concerted fashion, that is, the multiple sequences within a species have evolved in unison. The 3'-untranslated portion of the lysozyme genes appears to have escaped from concerted evolution due to inability to initiate concerted evolution, rather than due to reduced sequence similarity. The process of concerted evolution in stomach lysozymes may have had roles both in adapting lysozyme to the stomach environment in early ruminants as well as in retarding amino acid sequence evolution in the well adapted lysozyme of modern ruminants.

Animals↗
PubMed1990

DNA damage promotes jumping between templates during enzymatic amplification.

Pairs of templates and primers were designed so that only recombination events would lead to amplification via the polymerase chain reaction. This approach reveals that lesions such as breaks, apurinic sites, and UV damage in a DNA template can cause the extending primer to jump to another template during the polymerase chain reaction. By comparing sequences of amplification products that were determined directly or via bacterial cloning, it was shown that when the thermostable Thermus aquaticus DNA polymerase encounters the end of a template molecule, it sometimes inserts an adenosine residue; the prematurely terminated product then jumps to another template and polymerization continues, creating an in vitro recombination product. Consequently, amplification products from damaged templates such as archaeological DNA are made up of a high proportion of chimeric molecules. The illegitimate adenosine and thymidine residues in these molecules are detected when cloned molecules are sequenced, but are generally averaged out when the amplification product is sequenced directly. However, if site-specific lesions exist in template DNA or if the amplification is initiated from very few copies, direct sequencing also may yield incorrect sequences. The phenomenon of the "jumping polymerase chain reaction" can be exploited to assess the frequency and location of lesions in nucleic acids.

Adenosine↗